Highly Potent Acid Alpha-Glucosidase With Enhanced Carbohydrates

ABSTRACT

Recombinant human alpha glucosidase (rhGAA) composition derived from CHO cells that contains a more optimized glycan composition consisting of a higher amount of rhGAA containing N-glycans carrying mannose-6-phosphate (M6P) or bis-M6P than conventional rhGAAs, along with low amount of non-phosphorylated high mannose glycans, and low amount of terminal galactose on complex oligosaccharides. Compositions containing the rhGAA, and methods of use are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/249,175, filed Feb. 23, 2021, which is a division of U.S. patentapplication Ser. No. 16/252,505, filed Jan. 18, 2019 and issued as U.S.Pat. No. 10,961,522, which is a continuation of U.S. patent applicationSer. No. 15/515,808, filed Mar. 30, 2017 and issued as U.S. Pat. No.10,208,299, which is the U.S. national stage entry under 35 U.S.C. § 371of International Application No. PCT/US2015/053252, filed Sep. 30, 2015,which claims the benefit of priority to U.S. Provisional Application No.62/057,842, filed Sep. 30, 2014, U.S. Provisional Application No.62/057,847, filed Sep. 30, 2014, U.S. Provisional Application No.62/112,463, filed Feb. 5, 2015, and U.S. Provisional Application No.62/135,345, filed Mar. 19, 2015, each of which is incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:AMCS_005_07US_SeqList_ST25.txt, date recorded: Feb. 4, 2022, file size40,505 bytes).

BACKGROUND OF THE INVENTION Field of the Invention

The present invention involves the fields of medicine, genetics andrecombinant glycoprotein biochemistry, and, specifically, relates torecombinant human alpha glucosidase (rhGAA) compositions that have ahigher total content of mannose 6-phosphate-bearing glycans thatefficiently target CIMPR on muscle cells and subsequently deliver rhGAAto the lysosomes where it can break down abnormally high levels ofaccumulated glycogen. The rhGAA of the invention exhibits superiortargeting to muscle cells and subsequent delivery to lysosomes comparedto conventional rhGAA products and exhibits other pharmacokineticproperties that make it particularly effective for enzyme replacementtherapy of subjects having Pompe disease.

Description of the Related Art

Existing enzyme replacement therapies for Pompe Disease use conventionalrhGAA products that have a low total content of M6P and bis-M6P bearingglycans. Conventional Alglucosidase Alfa products are sold under thetrademarks LUMIZYME® and MYOZYME®. LUMIZYME® and MYOZYME® areconventional forms of rhGAA produced or marketed as biologics by Genzymeand approved by the U.S. Food and Drug Administration and are describedby reference to the Physician's Desk Reference (2014)(which is herebyincorporated by reference) or by their prescription labels approved foruse in the United States by the FDA as of Oct. 1, 2014. AlglucosidaseAlfa is identified as chemical name[199-arginine,223-histidine]prepro-α-glucosidase (human); molecularformula, C₄₇₅₈H₇₂₆₂N₁₂₇₄O₁₃₆₉S₃₅; CAS number 420794-05-0. These productsare administered to subjects with Pompe Disease, also known as glycogenstorage disease type II (GSD-II) or acid maltase deficiency disease.Enzyme replacement therapy seeks to treat Pompe Disease by replacing themissing GAA in lysosomes by administering rhGAA thus restoring theability of cell to break down lysosomal glycogen.

Pompe disease is an inherited lysosomal storage disease that resultsfrom a deficiency in acid α-glucosidase (GAA) activity. A person havingPompe Disease lacks or has reduced levels of acid alpha-glucosidase(GAA), the enzyme which breaks down glycogen, and a substance the bodyuses as an energy source. This enzyme deficiency causes excess glycogenaccumulation in the lysosomes, which are intra-cellular organellescontaining enzymes that ordinarily break down glycogen and othercellular debris or waste products. Glycogen accumulation in certaintissues of a subject having Pompe Disease, especially muscles, impairsthe ability of cells to function normally. In Pompe Disease, glycogen isnot properly metabolized and progressively accumulates in the lysosomes,especially in skeletal muscle cells and, in the infant onset form of thedisease, in cardiac muscle cells. The accumulation of glycogen damagesthe muscle and nerve cells as well as those in other affected tissues.

Traditionally, depending on the age of onset, Pompe disease isclinically recognized as either an early infantile form or as a lateonset form. The age of onset tends to parallel the severity of thegenetic mutation causing Pompe Disease. The most severe geneticmutations cause complete loss of GAA activity manifest as early onsetdisease during infancy. Genetic mutations that diminish GAA activity butdo not completely eliminate it are associated with forms of Pompedisease having delayed onset and progression. Infantile onset Pompedisease manifests shortly after birth and is characterized by muscularweakness, respiratory insufficiency and cardiac failure. Untreated, itis usually fatal within two years. Juvenile and adult onset Pompedisease manifest later in life and usually progress more slowly thaninfantile onset disease. This form of the disease, while it generallydoes not affect the heart, may also result in death, due to weakening ofskeletal muscles and those involved in respiration.

Current non-palliative treatment of Pompe disease involves enzymereplacement therapy (ERT) using recombinant human GAA (rhGAA) such asLUMIZYME® or MYOZYME®. The rhGAA is administered in an attempt toreplace or supplement the missing or defective GAA in a subject havingPompe Disease. However, since most of the rhGAA in conventional rhGAAproducts does not target muscle tissue it is non-productively eliminatedafter administration.

This occurs because conventional rhGAAs lack a high total content ofM6P- and bis-M6P-bearing glycans which target a rhGAA molecule to theCIMPR on target muscle cells where it is subsequently transported intothe cell's lysosomes. This cellular uptake of rhGAA for enzymereplacement therapy is facilitated by the specialized carbohydrate,mannose-6-phosphate (M6P), which binds to the cation-independent mannose6-phosphate receptor (CIMPR) present on cell surfaces for subsequentdelivery of the exogenous enzyme to lysosomes.

There are seven potential N-linked glycosylation sites on rhGAA. Sinceeach glycosylation site is heterogeneous in the type of N-linkedoligosaccharides (N-glycans) present, rhGAA consist of a complex mixtureof proteins with N-glycans having varying binding affinities for M6Preceptor and other carbohydrate receptors. rhGAA that contains a highmannose N-glycans having one M6P group (mono-M6P) binds to CIMPR withlow (˜6,000 nM) affinity while rhGAA that contains two M6P groups onsame N-glycan (bis-M6P) bind with high (˜2 nM) affinity. Representativestructures for non-phosphorylated, mono-M6P, and bis-M6P glycans areshown by FIG. 1A. The mannose-6-P group is shown by FIG. 1B. Once insidethe lysosome, rhGAA can enzymatically degrade accumulated glycogen.However, conventional rhGAAs have low total levels of M6P- andbis-M6P-bearing glycans and, thus, target muscle cells poorly resultingin inferior delivery of rhGAA to the lysosomes. The majority of rhGAAmolecules in these conventional products do not have phosphorylatedN-glycans, thereby lacking affinity for the CIMPR. Non-phosphorylatedhigh mannose glycans can also be cleared by the mannose receptor whichresults in non-productive clearance of the ERT (FIG. 2 ).

The other type of N-glycans, complex carbohydrates, which contain,galactose and sialic acids are also present on rhGAA. Since complexN-glycans are not phosphorylated they have no affinity for CIMPR.However, complex-type N-glycans with exposed galactose residues havemoderate to high affinity for the asialoglycoprotein receptor on liverhepatocytes which leads to rapid non-productive clearance of rhGAA (FIG.2 ).

The glycosylation of GAA or rhGAA can be enzymatically modified in vitroby the phosphotransferase and uncovering enzymes described by Canfield,et al., U.S. Pat. No. 6,534,300, to generate M6P groups. Enzymaticglycosylation cannot be adequately controlled and produces rhGAA havingundesirable immunological and pharmacological properties. Enzymaticallymodified rhGAA may contain only high-mannose N-glycans which all couldbe potentially enzymatically phosphorylated in vitro with aphosphotransferase/uncovering enzyme and may contain on average 5-6 M6Pgroups per GAA. The glycosylation patterns produced by in vitroenzymatic treatment of GAA are problematic because the additionalterminal mannose residues, particularly non-phosphorylated terminalmannose residues, negatively affect the pharmacokinetics of the modifiedrhGAA. When such an enzymatically modified product is administered invivo, these mannose groups increase non-productive clearance of the GAA,increase the uptake of the enzymatically-modified GAA by immune cells,and reduce rhGAA therapeutic efficacy due to less of the GAA reachingtargeted tissues, such as cardiac or skeletal muscle myocytes. Forexample, terminal non-phosphorylated mannose residues are known ligandsfor mannose receptors in the liver and spleen which leads to rapidclearance of the enzymatically-modified rhGAA and reduced targeting ofrhGAA to target tissue. Moreover, the glycosylation pattern ofenzymatically-modified GAA having high mannose N-glycans with terminalnon-phosphorylated mannose residues resembles that on glycoproteinsproduced in yeasts, molds and function increasing the risk of triggeringimmune or allergic responses, such as life-threatening severe allergic(anaphylactic) or hypersensitivity reactions, to the enzymaticallymodified rhGAA.

As explained above, conventional rhGAA products like LUMIZYME® have lowlevels of mono-phosphorylated glycans and even lower bis-phosphorylatedglycans. In order for a Pompe disease therapy to be efficacious rhGAAmust be delivered to the lysosomes in muscle cells. The low total amountof mono-M6P and bis-M6P targeting groups on conventional rhGAA limitscellular uptake via CIMPR and lysosomal delivery, thus makingconventional enzyme replacement therapy inefficient. For example, whileconventional rhGAA products at 20 mg/kg or higher doses do amelioratesome aspects of Pompe disease, they are not able to adequately reduceaccumulated glycogen in many target tissues, particularly skeletalmuscles to reverse disease progression.

Due to the inefficiency of delivering conventional enzyme replacementtherapies to lysosomes, such therapies are often associated with otherproblems, including generation of immune responses to GAA. A largeportion of the GAA in a conventional rhGAA does not contain glycansbearing mono- or bis-M6P, which target the rhGAA to muscle cells. Asubject's immune system is exposed to this excess non-phosphorylated GAAand can generate detrimental immune responses that recognize GAA.Induction of an immune responses to the non-phosphorylated GAA that doesnot enter the target tissues and deliver to the lysosomes increase therisk of treatment failure due to immunological inactivation of theadministered rhGAA and increases the risk of the patient experiencingdetrimental autoimmune or allergic reactions to the rhGAA treatment. TherhGAA according to the invention contains significantly less of thisnon-targeted, non-phosphorylated rhGAA, thus reducing exposure of apatient's immune system to it.

Logistically, larger doses impose additional burdens on the subject aswell as medical professionals treating the subject, such as lengtheningthe infusion time needed to administer rhGAA intravenously. This isbecause conventional rhGAA's contain a higher content ofnon-phosphorylated rhGAA which does not target the CIMPR on musclecells. rhGAA that does not bind to CIMPR on muscle cells and then enterthe lysosome does not enzymatically degrade glycogen there. Whenequivalent doses of a conventional rhGAA and the rhGAA according to theinvention are administered, more rhGAA in the composition according tothe invention binds CIMPR on muscle cells and then delivers to thelysosome. The rhGAA of the invention provides a doctor with the optionof administering a lower amount of rhGAA while delivering the same ormore rhGAA to the lysosome.

Current manufacturing processes used to make conventional rhGAA, such asMYOZYME® or LUMIZYME®, have not significantly increased the content ofM6P or bis-M6P because cellular carbohydrate processing is naturallycomplex and extremely difficult to manipulate. In view of thesedeficiencies of conventional rhGAA products, the inventors diligentlysought and identified ways to efficiently target rhGAA to muscle cellsand deliver it to the lysosome, minimize non-productive clearance ofrhGAA once administered, and thus more productively target rhGAA tomuscle tissue.

BRIEF SUMMARY OF THE INVENTION

In response to the problems associated with targeting and administeringconventional forms of rhGAA and to the difficulties associated withproducing such well-targeted forms of rhGAA, the inventors haveinvestigated and developed procedures for making rhGAA that moreefficiently targets the CIMPR and deliver it to lysosomes in muscletissues because it has a higher content of M6P- and bis-M6P glycan thanconventional rhGAA compositions. Moreover, rhGAA of the invention haswell-processed complex-type N-glycans which minimize non-productiveclearance of the rhGAA by non-target tissues.

Taking into account the problems associated with current enzymereplacement treatments using conventional rhGAA products such asLUMIZYME®, through diligent study and investigation the inventors havedeveloped a method for producing rhGAA in CHO cells having significantlyhigher total content of mono-M6P and bis-M6P glycans which target CIMPRon muscle cells and then deliver the rhGAA to the lysosomes.

The rhGAA produced by this method also has advantageous pharmacokineticproperties by virtue of its overall glycosylation pattern that increasestarget tissue uptake and decreases non-productive clearance followingadministration to a subject having Pompe Disease. The inventors showthat the rhGAA of the invention, as exemplified by rhGAA designated asATB-200, is more potent in and more efficient at targeting skeletalmuscle tissues than conventional rhGAA such as LUMIZYME®. The rhGAAaccording to the invention has a superior ability to productively targetmuscle tissues in patients having Pompe Disease and reducenon-productive clearance of rhGAA as illustrated by FIG. 2 .

The superior rhGAA according to the invention may be further completedor combined with chaperones or conjugated to other groups that targetthe CIMPR in muscle tissue, such as portions of IGF2 that bind to thisreceptor. The Examples below show that the rhGAA of the invention,exemplified by ATB-200 rhGAA, exceeds the existing standard of care forenzyme replacement therapy by providing significantly better glycogenclearance in skeletal muscle as compared to existing regimen using theconventional rhGAA product LUMIZYME®.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a non-phosphorylated high mannose glycan, a mono-M6Pglycan, and a bis-M6P glycan. FIG. 1B shows the chemical structure ofthe M6P group.

FIG. 2A describes productive targeting of rhGAA via glycans bearing M6Pto target tissues (e.g., muscle tissues of subject with Pompe Disease).FIG. 2B describes non-productive drug clearance to non-target tissues(e.g., liver and spleen) or by binding of non-M6P glycans to non-targettissues.

FIG. 3A graphically depicts a CIMPR receptor (also known as an IGF2receptor) and domains of this receptor. FIG. 3B is a table showingbinding affinity (nMolar) of glycans bearing bis- and mono-M6P forCIMPR, the binding affinity of high mannose-type glycans to mannosereceptors, and the binding affinity of de-sialyated complex glycan forasialyoglycoprotein receptors. RhGAA that has glycans bearing M6P andbis-M6P can productively bind to CIMPR on muscle target cells. RhGAAthat has high mannose glycans and de-sialylated glycans cannon-productively bind to non-target cells bearing the correspondingreceptors.

FIGS. 4A and 4B, respectively, show the results of CIMPR affinitychromatography of LUMIZYME® and MYOZYME®. The dashed lines refer to theM6P elution gradient. Elution with M6P displaces GAA molecules bound viaan M6P-containing glycan to CIMPR. As shown in FIG. 4A, 78% of the GAAactivity in LUMIZYME® eluted prior to addition of M6P. FIG. 4B showsthat 73% of the GAA MYOZYME® activity eluted prior to addition of M6P.Only 22% or 27% of the rhGAA in LUMIZYME® or MYOZYME®, respectively, waseluted with M6P. These figures show that most of the rhGAA in these twoconventional rhGAA products lack glycans having M6P needed to targetCIMPR in target muscle tissues.

FIG. 5 . shows a DNA construct for transforming CHO cells with DNAencoding rhGAA. CHO cells were transformed with a DNA construct encodingrhGAA (SEQ ID NO: 2).

FIGS. 6A and 6B show the results of CIMPR affinity chromatography ofLUMIZYME® and ATB-200 rhGAA. As apparent from FIG. 6B, about 70% of therhGAA in ATB-200 rhGAA contained M6P.

FIGS. 7A-7B show ATB-200 rhGAA purification, Embodiments 1 & 2.

FIG. 8 shows Polywax elution profiles of LUMIZYME® and ATB-200 rhGAAs.

FIG. 9 shows a summary of N-glycan structures of LUMIZYME® compared tothree different preparations of ATB200 rhGAA, identified as BP-rhGAA,ATB200-1 and ATB200-2.

FIG. 10A compares the CIMPR binding affinity of ATB-200 rhGAA (lefttrace) with that of LUMIZYME® (right trace). FIG. 10B describes theBis-M6P content of LUMIZYME® and ATB-200 rhGAA.

FIG. 11A compares ATB-200 rhGAA activity (left trace; diamonds) withLUMIZYME® rhGAA activity (right trace; circles) inside normalfibroblasts at various GAA concentrations, and shows ATB-200 rhGAAactivity (triangle) compared to LUMIZYME® activity (square) in thepresence of M6P. FIG. 11B compares ATB-200 rhGAA activity (left trace;diamonds) with LUMIZYME® rhGAA activity (right trace; circles) insidefibroblasts from a subject having Pompe Disease at various GAAconcentrations, and shows ATB-200 rhGAA activity (triangle) compared toLUMIZYME® activity (square) in the presence of M6P. FIG. 11C compares(K_(uptake)) of fibroblasts from normal subjects and subjects with PompeDisease.

FIG. 12A shows the amount of glycogen relative to protein in heartmuscle after contact with vehicle (negative control), with 20 mg/mlLUMIZYME®, or with 5, 10 or 20 mg/kg ATB-200 rhGAA. FIG. 12B shows theamount of glycogen relative to protein in quadriceps muscle aftercontact with vehicle (negative control), with 20 mg/ml LUMIZYME®, orwith 5, 10 or 20 mg/kg ATB-200 rhGAA. FIG. 12C shows the amount ofglycogen relative to protein in triceps muscle after contact withvehicle (negative control), with 20 mg/ml LUMIZYME®, or with 5, 10 or 20mg/kg ATB-200 rhGAA. ATB-200 rhGAA produced significant glycogenreductions in quadriceps and triceps muscle compared to the negativecontrol and compared to LUMIZYME®.

FIGS. 13A-13B show that ATB-200 rhGAA stability is improved in thepresence of chaperone AT2221. The first, left trace in FIG. 13A showspercentage of unfolded ATB-200 rhGAA protein at various temperatures atpH 7.4 (blood pH). The last, right trace shows percentage of unfoldedATB-200 rhGAA protein at various temperatures at pH 5.2 (lysosome pH).The three intermediate traces show the effects of 10 μg, 30 μg, or 100μg of AT2221 chaperone on protein folding. These data show that AT2221prevents unfolding of ATB-200 rhGAA at blood pH compared to the controlsample. The improvement of Tm at neutral pH by AT2221 is summarized inFIG. 13B.

FIG. 14 shows that the combination of ATB-200 rhGAA and chaperone AT2221provided significantly better glycogen clearance in GAA knock-out micethan treatments with LUMIZYME® and AT2221 or controls of eitherLUMIZYME® or ATB200 rhGAAs without the AT2221 chaperone.

FIG. 15 shows residual glycogen in quadriceps muscle after treatmentwith LUMIZYME®, ATB-200 rhGAA, or ATB-200 rhGAA and variousconcentrations of the AT2221 chaperone.

FIGS. 16A-16D show improvement of Skeletal Muscle Pathology in Micetreated with ATB200+Miglustat (AT2221) over those treated with ERTalone. PAS glycogen staining (FIG. 16A) and EM (FIG. 16B) of muscletissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAAand miglustat (AT-2221). FIG. 16C: Evaluation of lysosomal proliferationby LAMP-1 marker. FIG. 16D: Identification of Type I and Type II musclefibers.

FIGS. 17A-17B show improvement of Skeletal Muscle Pathology in Micetreated with ATB-200+Miglustat (AT2221) over those treated with ERTalone. PAS glycogen staining (FIG. 17A) of muscle tissue from GAA KOmice treated with conventional rhGAA or ATB-200 rhGAA and miglustat(AT-2221). FIG. 17B: Evaluation of lysosomal proliferation by LAMP-1marker.

DETAILED DESCRIPTION OF THE INVENTION

Definitions: The terms used in this specification generally have theirordinary meanings in the art, within the context of this invention andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the compositionsand methods of the invention and how to make and use them.

The term “GAA” refers to human acid α-glucosidase (GAA) an enzyme thatcatalyzes the hydrolysis of α-1,4- and α-1,6-glycosidic linkages oflysosomal glycogen as well as to insertional, relational or substitutionvariants of the GAA amino acid sequence and fragments of a longer GAAsequence that exert enzymatic activity. The term “rhGAA” is used todistinguish endogenous GAA from synthetic or recombinant-produced GAA,such as that produced by transformation of CHO cells with DNA encodingGAA. An exemplary DNA sequence encoding GAA is SEQ ID NO: 2, which isincorporated by reference. GAA and rhGAA may be present in a compositioncontaining a mixture of GAA molecules having different glycosylationpatterns, such as a mixture of rhGAA molecules bearing mono-M6P orbis-M6P groups on their glycans and GAA molecules that do not bear M6Por bis-M6P. GAA and rhGAA may also be completed with other compounds,such as chaperones, or may be bound to other moieties in a GAA or rhGAAconjugate, such as bound to an IGF2 moiety that targets the conjugate toCIMPR and subsequently delivers it to the lysosomes.

A “subject” or “patient” is preferably a human, though other mammals andnon-human animals having disorders involving accumulation of glycogenmay also be treated. A subject may be a fetus, a neonate, child,juvenile or an adult with Pompe disease or other glycogen storage oraccumulation disorder. One example of an individual being treated is anindividual (fetus, neonate, child, juvenile, adolescent, or adult human)having GSD-II (e.g., infantile GSD-II, juvenile GSD-II, or adult-onsetGSD-II). The individual can have residual GAA activity, or no measurableactivity. For example, the individual having GSD-II can have GAAactivity that is less than about 1% of normal GAA activity (infantileGSD-II), GAA activity that is about 1-10% of normal GAA activity(juvenile GSD-II), or GAA activity that is about 10-40% of normal GAAactivity (adult GSD-II).

The terms, “treat” and “treatment,” as used herein, refer toamelioration of one or more symptoms associated with the disease,prevention or delay of the onset of one or more symptoms of the disease,and/or lessening of the severity or frequency of one or more symptoms ofthe disease. For example, treatment can refer to improvement of cardiacstatus (e.g., increase of end-diastolic and/or end-systolic volumes, orreduction, amelioration or prevention of the progressive cardiomyopathythat is typically found in GSD-II) or of pulmonary function (e.g.,increase in crying vital capacity over baseline capacity, and/ornormalization of oxygen desaturation during crying); improvement inneurodevelopment and/or motor skills (e.g., increase in AIMS score);reduction of glycogen levels in tissue of the individual affected by thedisease; or any combination of these effects. In one preferredembodiment, treatment includes improvement of cardiac status,particularly in reduction or prevention of GSD-II-associatedcardiomyopathy.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a baseline measurement, such as ameasurement in the same individual prior to initiation of the treatmentdescribed herein, or a measurement in a control individual (or multiplecontrol individuals) in the absence of the treatment described herein. Acontrol individual is an individual afflicted with the same form ofGSD-II (either infantile, juvenile or adult-onset) as the individualbeing treated, who is about the same age as the individual being treated(to ensure that the stages of the disease in the treated individual andthe control individual(s) are comparable).

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 95% pure; morepreferably, at least 97% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, enzymatic assay andother methods known in the art. In a specific embodiment, purified meansthat the level of contaminants is below a level acceptable to regulatoryauthorities for safe administration to a human or non-human animal.Recombinant proteins may be isolated or purified from CHO cells usingmethods known in the art including by chromatographic size separation,affinity chromatography or anionic exchange chromatography.

The term “genetically modified” or “recombinant” refers to cells, suchas CHO cells, that express a particular gene product, such as rhGAA orATB-200 rhGAA, following introduction of a nucleic acid comprising acoding sequence which encodes the gene product, along with regulatoryelements that control expression of the coding sequence. Introduction ofthe nucleic acid may be accomplished by any method known in the artincluding gene targeting and homologous recombination. As used herein,the term also includes cells that have been engineered to express oroverexpress an endogenous gene or gene product not normally expressed bysuch cell, e.g., by gene activation technology.

“Pompe Disease” refers to an autosomal recessive LSD characterized bydeficient acid alpha glucosidase (GAA) activity which impairs lysosomalglycogen metabolism. The enzyme deficiency leads to lysosomal glycogenaccumulation and results in progressive skeletal muscle weakness,reduced cardiac function, respiratory insufficiency, and/or CNSimpairment at late stages of disease. Genetic mutations in the GAA generesult in either lower expression or produce mutant forms of the enzymewith altered stability, and/or biological activity ultimately leading todisease. (see generally Hirschhorn R, 1995, Glycogen Storage DiseaseType II: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic andMolecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill,New York, 7th ed., pages 2443-2464). The three recognized clinical formsof Pompe Disease (infantile, juvenile and adult) are correlated with thelevel of residual α-glucosidase activity (Reuser A J et al., 1995,Glycogenosis Type II (Acid Maltase Deficiency), Muscle & NerveSupplement 3, S61-S69). Infantile Pompe disease (type I or A) is mostcommon and most severe, characterized by failure to thrive, generalizedhypotonic, cardiac hypertrophy, and cardiorespiratory failure within thesecond year of life. Juvenile Pompe disease (type II or B) isintermediate in severity and is characterized by a predominance ofmuscular symptoms without cardiomegaly. Juvenile Pompe individualsusually die before reaching 20 years of age due to respiratory failure.Adult Pompe disease (type III or C) often presents as a slowlyprogressive myopathy in the teenage years or as late as the sixth decade(Felicia K J et al., 1995, Clinical Variability in Adult-Onset AcidMaltase Deficiency: Report of Affected Sibs and Review of theLiterature, Medicine 74, 131-135). In Pompe, it has been shown thatα-glucosidase is extensively modified post-translationally byglycosylation, phosphorylation, and proteolytic processing. Conversionof the 110 kilo Dalton (kids) precursor to 76 and 70 kids mature formsby proteolysis in the lysosome is required for optimum glycogencatalysis. As used herein, the term “Pompe Disease” refers to all typesof Pompe Disease. The formulations and dosing regimens disclosed in thisapplication may be used to treat, for example, Type I, Type II or TypeIII Pompe Disease.

Non-Limiting Embodiments of the Invention

A rhGAA composition derived from CHO cells that contains a higher amountof rhGAA containing N-glycans carrying mono-mannose-6-phosphate (M6P) orbis-M6P than conventional rhGAA as exemplified by LUMIZYME®. Anexemplary rhGAA composition according to the invention is ATB-200(sometimes designated ATB-200, ATB-200 or CBP-rhGAA) which is describedin the Examples. The rhGAA of the invention (ATB-200) has been shown tobind the CIMPR with high affinity (K_(D)˜2-4 nM) and to be efficientlyinternalized by Pompe fibroblasts and skeletal muscle myoblasts(K_(uptake)˜7-14 nM). ATB-200 was characterized in vivo and shown tohave a shorter apparent plasma half-life (t_(1/2)˜45 min) than thecurrent rhGAA ERT (t_(1/2)˜60 min).

The amino acid sequence of the rhGAA can be at least 70%, 75%, 80%, 85%,95% or 99% identical, or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredeletions, substitutions or additions to the amino acid sequencedescribed by SEQ ID NO: 1, 3 or 4. In some embodiments of the GAA orrhGAA of the invention, such as in ATB-200 rhGAA, the GAA or rhGAA willcomprise a wild-type GAA amino acid sequence such as that of SEQ ID NO:1 or 3. In other non-limiting embodiments, the rhGAA comprises a subsetof the amino acid residues present in a wild-type GAA, wherein thesubset includes the amino acid residues of the wild-type GAA that formthe active site for substrate binding and/or substrate reduction. In oneembodiment, the rhGAA is glucosidase alfa, which is the human enzymeacid a-glucosidase (GAA), encoded by the most predominant of nineobserved haplotypes of this gene. The rhGAA of the invention, includingATB-200 rhGAA, may comprise an amino acid sequence that is 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of humanalpha glucosidase, such as that given by accession number AHE24104.1(GI:568760974)(SEQ ID NO: 1) and which is incorporated by reference toU.S. Pat. No. 8,592,362 or to the amino acid sequence of NP 000143.2(SEQ ID NO: 4). A nucleotide and amino acid sequence for GAA is alsogiven by SEQ ID NOS: 2 and 3, respectively. Variants of this amino acidsequence also include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more amino acid deletions, insertions or substitutions to the GAAamino acid sequence below. Polynucleotide sequences encoding GAA andsuch variant human GAAs are also contemplated and may be used torecombinantly express rhGAAs according to the invention.

Various alignment algorithms and/or programs may be used to calculatethe identity between two sequences, including FASTA, or BLAST which areavailable as a part of the GCG sequence analysis package (University ofWisconsin, Madison, Wis.), and can be used with, e.g., default setting.For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99%identity to specific polypeptides described herein and preferablyexhibiting substantially the same functions, as well as polynucleotideencoding such polypeptides, are contemplated. Unless otherwise indicateda similarity score will be based on use of BLOSUM62. When BLASTP isused, the percent similarity is based on the BLASTP positives score andthe percent sequence identity is based on the BLASTP identities score.BLASTP “Identities” shows the number and fraction of total residues inthe high scoring sequence pairs which are identical; and BLASTP“Positives” shows the number and fraction of residues for which thealignment scores have positive values and which are similar to eachother. Amino acid sequences having these degrees of identity orsimilarity or any intermediate degree of identity of similarity to theamino acid sequences disclosed herein are contemplated and encompassedby this disclosure. The polynucleotide sequences of similar polypeptidesare deduced using the genetic code and may be obtained by conventionalmeans, in particular by reverse translating its amino acid sequenceusing the genetic code.

Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10,or 5% of the total rhGAA in the composition according to the inventionlacks an N-glycan bearing M6P or bis-M6P or lacks a capacity to bind tothe cationic independent mannose-6-phosphate receptor (CIMPR).Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,99%, <100% or more of the rhGAA in the composition comprises at leastone N-glycan bearing M6P and/or bis-M6P or has the capacity to bind toCIMPR.

The rhGAA molecules in the rhGAA composition of the invention may have1, 2, 3 or 4 M6P groups on their glycans. For example, only one N-glycanon an rhGAA molecule may bear M6P (mono-phosphorylated), a singleN-glycan may bear two M6P groups (bis-phosphorylated), or two differentN-glycans on the same rhGAA molecule may bear single M6P groups. rhGAAmolecules in the rhGAA composition may also have N-glycans bearing noM6P groups. In another embodiment, on average the N-glycans containgreater than 3 mol/mol of M6P and greater than 4 mol/mol sialic acid. Onaverage at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycanson the rhGAA may be in the form of a mono-M6P glycan, for example, about6.25% of the total glycans may carry a single M6P group and on average,at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans on therhGAA are in the form of a bis-M6P glycan and on average less than 25%of total rhGAA of the invention contains no phosphorylated glycanbinding to CIMPR.

The rhGAA composition according to the invention may have an averagecontent of N-glycans carrying M6P ranging from 0.5 to 7.0 mol/mol rhGAAor any intermediate value of subrange including 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mol/mol rhGAA. As shownin the Examples, the rhGAA of the invention can be fractionated toprovide rhGAA compositions with different average numbers of M6P-bearingor bis-M6P-bearing glycans on the rhGAA thus permitting furthercustomization of rhGAA targeting to the lysosomes in target tissues byselecting a particular fraction or by selectively combining differentfractions.

Up to 60% of the N-glycans on the rhGAA may be fully sialyated, forexample, up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans may befully sialyated. In some embodiments from 4 to 20% of the totalN-glycans in the rhGAA composition are fully sialylated.

In other embodiments no more than 5%, 10%, 20% or 30% of N-glycans onthe rhGAA carry sialic acid and a terminal Gal. This ranges includes allintermediate values and subranges, for example, 7 to 30% of the totalN-glycans on the rhGAA in the composition can carry sialic acid andterminal Gal.

In yet other embodiments, no more than 5, 10, 15, 16, 17, 18, 19 or 20%of the N-glycans on the rhGAA have a terminal Gal only and do notcontain sialic acid. This range includes all intermediate values andsubranges, for example, from 8 to 19% of the total N-glycans on therhGAA in the composition may have terminal Gal only and do not containsialic acid.

In other embodiments of the invention 40, 45, 50, 55 to 60% of the totalN-glycans on the rhGAA in the composition are complex type N-glycans; orno more than 1, 2, 3, 4, 5, 6, 7% of total N-glycans on the rhGAA in thecomposition are hybrid-type N-glycans; no more than 5, 10, or 15% of thehigh mannose-type N-glycans on the rhGAA in the composition arenon-phosphorylated; at least 5% or 10% of the high mannose-typeN-glycans on the rhGAA in the composition are mono-M6P phosphorylated;and/or at least 1 or 2% of the high mannose-type N-glycans on the rhGAAin the composition are bis-M6P phosphorylated. These values include allintermediate values and subranges. An rhGAA composition according to theinvention may meet one or more of the content ranges described above.

In some embodiments, the rhGAA composition of the invention will bear,on average, 2.0 to 8.0 sialic acid residues per mol of rhGAA. This rangeincludes all intermediate values and subranges including 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 residues/mol rhGAA.Sialic acid residues may prevent non-productive clearance byasialoglycoprotein receptors.

The rhGAA composition of the invention is preferably produced by CHOcells, such as CHO cell line GA-ATB-200, or by a subculture orderivative of such a CHO cell culture. DNA constructs, which expressallelic variants of GAA or other variant GAA amino acid sequences suchas those that are at least 90%, 95% or 99% identical to SEQ ID NO: 1,may be constructed and expressed in CHO cells. Those of skill in the artcan select alternative vectors suitable for transforming CHO cells forproduction of such DNA constructs.

The inventors have found that rhGAA having superior ability to targetthe CIMPR and cellular lysosomes as well as glycosylation patterns thatreduce its non-productive clearance in vivo can be produced usingChinese hamster ovary (CHO) cells. These cells can be induced to expressrhGAA with significantly higher levels of total M6P and bis-M6P thanconventional rhGAA products. The recombinant human GAA produced by thesecells, for example, as exemplified by rhGAA ATB-200 described in theExamples, has significantly more muscle cell-targeting M6P and bis-M6Pgroups than conventional GAA, such as LUMIZYME® and has been shown toefficiently bind to CIMPR and be efficiently taken up by skeletal muscleand cardiac muscle. It has also been shown to have a glycosylationpattern that provides a favorable pharmacokinetic profile and reducesnon-productive clearance in vivo.

The rhGAA according to the invention may be formulated as apharmaceutical composition or used in the manufacture of a medicamentfor treatment of Pompe Disease or other conditions associated with adeficient of GAA. The compositions can be formulated with aphysiologically acceptable carrier or excipient. The carrier andcomposition can be sterile and otherwise suit the mode ofadministration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well ascombinations thereof. The pharmaceutical preparations can, if desired,be mixed with auxiliary agents, e.g., surfactants, such as polysorbateslike polysorbate 80, lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. In a preferredembodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,magnesium carbonate, etc. In a preferred embodiment the rhGAA isadministered by IV infusion.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration is a solution in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic to ease pain at thesite of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage faun, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampule or sachet indicating the quantity of activeagent. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water, saline or dextrose/water. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The rhGAA can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

rhGAA (or a composition or medicament containing GAA) is administered byan appropriate route. In one embodiment, the GAA is administeredintravenously. In other embodiments, GAA is administered by directadministration to a target tissue, such as to heart or skeletal muscle(e.g., intramuscular), or nervous system (e.g., direct injection intothe brain; intraventricularly; intrathecally). More than one route canbe used concurrently, if desired.

The rhGAA (or a composition or medicament containing GAA) isadministered in a therapeutically effective amount (e.g., a dosageamount that, when administered at regular intervals, is sufficient totreat the disease, such as by ameliorating symptoms associated with thedisease, preventing or delaying the onset of the disease, and/or alsolessening the severity or frequency of symptoms of the disease, asdescribed above). The amount which will be therapeutically effective inthe treatment of the disease will depend on the nature and extent of thedisease's effects, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed will also depend on the route of administration, and theseriousness of the disease, and should be decided according to thejudgment of a practitioner and each patient's circumstances. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems. In a preferred embodiment, thetherapeutically effective amount is equal of less than 20 mg enzyme/kgbody weight of the individual, preferably in the range of about 1-10 mgenzyme/kg body weight, and even more preferably about 10 mg enzyme/kgbody weight or about 5 mg enzyme/kg body weight. The effective dose fora particular individual can be varied (e.g., increased or decreased)over time, depending on the needs of the individual. For example, intimes of physical illness or stress, or if anti-GAA antibodies becomepresent or increase, or if disease symptoms worsen, the amount can beincreased.

The therapeutically effective amount of GAA (or composition ormedicament containing GAA) is administered at regular intervals,depending on the nature and extent of the disease's effects, and on anongoing basis. Administration at a “regular interval,” as used herein,indicates that the therapeutically effective amount is administeredperiodically (as distinguished from a one-time dose). The interval canbe determined by standard clinical techniques. In preferred embodiments,GAA is administered monthly, bimonthly; weekly; twice weekly; or daily.The administration interval for a single individual need not be a fixedinterval, but can be varied over time, depending on the needs of theindividual. For example, in times of physical illness or stress, ifanti-GAA antibodies become present or increase, or if disease symptomsworsen, the interval between doses can be decreased. In someembodiments, a therapeutically effective amount of 5, 10, 20, 50, 100,or 200 mg enzyme/kg body weight is administered twice a week, weekly orevery other week with or without a chaperone.

The GAA or rhGAA of the invention may be prepared for later use, such asin a unit dose vial or syringe, or in a bottle or bag for intravenousadministration. Kits containing the GAA or rhGAA, as well as optionalexcipients or other active ingredients, such as chaperones or otherdrugs, may be enclosed in packaging material and accompanied byinstructions for reconstitution, dilution or dosing for treating asubject in need of treatment, such as a patient having Pompe disease.

GAA (or a composition or medicament containing GAA) can be administeredalone, or in conjunction with other agents, such as a chaperone. rhGAAwith different degrees of glycosylation with mono-M6P or bis-M6P may beadministered or combinations of rhGAAs with different degrees of M6P orbisM6P glycosylate administered.

In some embodiments the rhGAA composition of the invention will becomplexed or admixed with a chaperone, such as AT-2220 or AT-2221.Chaperones, sometimes referred to as “pharmacological chaperones,” arecompounds that when complexed or coadministered with rhGAA modify itspharmacokinetics and other pharmacological properties. Representativechaperones exemplified herein include AT2221 (miglustat,N-butyl-deoxynojirimycin) and AT2220 (duvoglustat HCl,1-deoxynojirimycin). Such complexing or admixing may occur outside thebody or inside the body, for example, where separate dosages of therhGAA and chaperone are administered. For example, targeting of activerhGAA, its fractions, or derivatives of the invention to CIMPR andsubsequently to cellular lysosomes may be improved by combining itduvoglustat-HCl (AT2220, deoxynojirimycine, AT2220) or miglustat(AT2221, N-butyl-deoxynojirimycin). The Examples below show significantglycogen substrate reductions in key skeletal muscles of GAA-knock-outmice receiving the well-targeted rhGAA of the invention in combinationwith a chaperone.

Another aspect of the invention pertains to CHO cells or theirderivatives or other equivalents that produce the rhGAA according to theinvention. One example of such a CHO cell line is GA-ATB-200 or asubculture thereof that produces a rhGAA composition as describedherein. Such CHO cell lines may contain multiple copies of a gene, suchas 5, 10, 15, or 20 or more copies, of a polynucleotide encoding GAA.

The high M6P and bis-M6P rhGAA of the invention, such as ATB-200 rhGAA,can be produced by transforming CHO cells (Chinese hamster ovary cells)with a DNA construct that encodes GAA. While CHO cells have beenpreviously used to make rhGAA, it was not appreciated that transformedCHO cells could be cultured and selected in a way that would producerhGAA having a high content of M6P and bis-M6P glycans which target theCIMPR.

Surprisingly, the inventors found that it was possible to transform CHOcell lines, select transformants that produce rhGAA containing a highcontent of glycans bearing M6P or bis-M6P that target the CIMPR, and tostably express this high-M6P rhGAA. Thus, a related aspect of theinvention is directed to method for making these CHO cell lines. Thismethod involves transforming a CHO cell with DNA encoding GAA or a GAAvariant, selecting a CHO cell that stably integrates the DNA encodingGAA into its chromosome(s) and that stably expresses GAA, and selectinga CHO cell that expresses GAA having a high content of glycans bearingM6P or bis-M6P, and, optionally, selecting a CHO cell having N-glycanswith high sialic acid content and/or having N-glycans with a lownon-phosphorylated high-mannose content.

These CHO cell lines may be used to produce rhGAA and rhGAA compositionsaccording to the invention by culturing the CHO cell line and recoveringsaid composition from the culture of CHO cells.

The rhGAA composition of the invention or its fractions or derivativesis advantageously used to treat subjects having a condition, disorder ordisease associated with insufficient lysosomal GAA by administering therhGAA composition. A subject in need of treatment includes those havingGlycogen Storage Disease Type II (Pompe Disease) as well as otherconditions, disorders or diseases which would benefit from theadministration of the rhGAA.

The Examples below show that the rhGAA of the invention (ATB-200) istaken up by skeletal muscle cells, binds to CIMPR and effectivelyremoves glycogen from skeletal muscle cells when administered at asignificantly lower dosage than conventional rhGAA products. A reductionof up to 75% of glycogen in skeletal muscle myoblast was attained inGAA-knockout mice using a biweekly regimen of intravenous administrationof ATB-200. These reductions exceeded those provided by the same amountof LUMIZYME® showing that the rhGAA of the invention, which has anenhanced content of N-glycans bearing M6P and bis-M6P, provided superiorreductions in glycogen substrate. Due to the improved targeting,pharmacodynamics and pharmacokinetics of the rhGAA composition of theinvention may be administered in a lower dosage than conventional rhGAAproducts such as LUMIZYME® or MYOZYME®.

It may be used to degrade, decrease or remove glycogen from cardiacmuscle, smooth muscle, or striated muscle. Examples of skeletal orstriated muscles subject to treatment include at least one muscleselected from the group consisting of abductor digiti minimi (foot),abductor digiti minimi (hand), abductor halluces, abductor pollicisbrevis, abductor pollicis longus, adductor brevis, adductor halluces,adductor longus, adductor magnus, adductor pollicis, anconeus,articularis cubiti, articularis genu, aryepiglotticus, aryjordanicus,auricularis, biceps brachii, biceps femoris, brachialis,brachioradialis, buccinators, bulbospongiosus, constrictor ofpharynx—inferior, constrictor of pharynx-middle, constrictor ofpharynx—superior, coracobrachialis, corrugator supercilii, cremaster,cricothyroid, dartos, deep transverse perinei, deltoid, depressor angulioris, depressor labii inferioris, diaphragm, digastric, digastric(anterior view), erector spinae—spinalis, erector spinae—iliocostalis,erector spinae—longissimus, extensor carpi radialis brevis, extensorcarpi radialis longus, extensor carpi ulnaris, extensor digiti minimi(hand), extensor digitorum (hand), extensor digitorum brevis (foot),extensor digitorum longus (foot), extensor hallucis longus, extensorindicis, extensor pollicis brevis, extensor pollicis longus, externaloblique abdominis, flexor carpi radialis, flexor carpi ulnaris, flexordigiti minimi brevis (foot), flexor digiti minimi brevis (hand), flexordigitorum brevis, flexor digitorum longus (foot), flexor digitorumprofundus, flexor digitorum superficialis, flexor hallucis brevis,flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus,frontalis, gastrocnemius, gemellus inferior, gemellus superior,genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteusminimus, gracilis, hyoglossus, iliacus, inferior oblique, inferiorrectus, infraspinatus, intercostals external, intercostals innermost,intercostals internal, internal oblique abdominis, interossei-dorsal ofhand, interossei-dorsal of foot, interossei-palmar of hand,interossei-plantar of foot, interspinales, intertransversarii, intrinsicmuscles of tongue, ishiocavernosus, lateral cricoarytenoid, lateralpterygoid, lateral rectus, latissimus dorsi, levator anguli oris,levator ani-coccygeus, levator ani—iliococcygeus, levatorani-pubococcygeus, levator ani-puborectalis, levator ani-pubovaginalis,levator labii superioris, levator labii superioris, alaeque nasi,levator palpebrae superioris, levator scapulae, levator veli palatine,levatores costarum, longus capitis, longus colli, lumbricals of foot,lumbricals of hand, masseter, medial pterygoid, medial rectus, mentalis,m. uvulae, mylohyoid, nasalis, oblique arytenoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus (A), obturator internus (B), omohyoid, opponens digiti minimi(hand), opponens pollicis, orbicularis oculi, orbicularis oris,palatoglossus, palatopharyngeus, palmaris brevis, palmaris longus,pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneuslongus, peroneus tertius, piriformis (A), piriformis (B), plantaris,platysma, popliteus, posterior cricoarytenoid, procerus, pronatorquadratus, pronator teres, psoas major, psoas minor, pyramidalis,quadratus femoris, quadratus lumborum, quadratus plantae, rectusabdominis, rectus capitus anterior, rectus capitus lateralis, rectuscapitus posterior major, rectus capitus posterior minor, rectus femoris,rhomboid major, rhomboid minor, risorius, salpingopharyngeus, sartorius,scalenus anterior, scalenus medius, scalenus minimus, scalenusposterior, semimembranosus, semitendinosus, serratus anterior, serratusposterior inferior, serratus posterior superior, soleus, sphincter ani,sphincter urethrae, splenius capitis, splenius cervicis, stapedius,sternocleidomastoid, sternohyoid, sternothyroid, styloglossus,stylohyoid, stylohyoid (anterior view), stylopharyngeus, subclavius,subcostalis, subscapularis, superficial transverse, perinei, superioroblique, superior rectus, supinator, supraspinatus, temporalis,temporoparietalis, tensor fasciae lata, tensor tympani, tensor velipalatine, teres major, teres minor, thyro-arytenoid & vocalis,thyro-epiglotticus, thyrohyoid, tibialis anterior, tibialis posterior,transverse arytenoid, transversospinalis—multifidus,transversospinalis—rotatores, transversospinalis—semispinalis,transversus abdominis, transversus thoracis, trapezius, triceps, vastusintermedius, vastus lateralis, vastus medialis, zygomaticus major, andzygomaticus minor.

The GAA composition of the invention may also be administered to, orused to treat, type 1 (slow twitch) muscle fiber or type 2 (fast twitch)muscle fiber or subjects accumulating glycogen in such muscle fibers.Type I, slow twitch, or “red” muscle, is dense with capillaries and isrich in mitochondria and myoglobin, giving the muscle tissue itscharacteristic red color. It can carry more oxygen and sustain aerobicactivity using fats or carbohydrates as fuel. Slow twitch fiberscontract for long periods of time but with little force. Type II, fasttwitch muscle, has three major subtypes (IIa, IIx, and IIb) that vary inboth contractile speed and force generated. Fast twitch fibers contractquickly and powerfully but fatigue very rapidly, sustaining only short,anaerobic bursts of activity before muscle contraction becomes painful.They contribute most to muscle strength and have greater potential forincrease in mass. Type IIb is anaerobic, glycolytic, “white” muscle thatis least dense in mitochondria and myoglobin. In small animals (e.g.,rodents) this is the major fast muscle type, explaining the pale colorof their flesh.

The rhGAA composition of the invention, its fractions or derivatives maybe administered systemically, for example, by intravenous (IV) infusion,or administered directly into a desired site, such as into cardiac orskeletal muscle, such as quadriceps, triceps, or diaphragm. It may beadministered to myocytes, particular muscle tissues, muscles, or musclegroups. For example, such a treatment may administer intramuscularly therhGAA composition directly into a subject's quadriceps or triceps ordiaphragm.

As mentioned above, the rhGAA composition of the invention, itsfractions or derivatives can be complexed or admixed with a chaperone,such as AT-2220 (Duvoglustat HCl, 1-Deoxynojirimycin) orAT-2221(Miglustat, N-butyl-deoxynojirimycin) or their salts to improvethe pharmacokinetics of the rhGAA administration. The rhGAA and thechaperone may be administered together or separately. When administeredsimultaneously the GAA in the composition may be preloaded with thechaperone. Alternatively, the GAA and the chaperone may be administeredseparately either at the same time or at different times.

Representative dosages of AT2221 range from 0.25 to 400 mg/kg,preferably from 0.5-200 mg/kg, and most preferably from 2 to 50 mg/kg.Specific dosages of AT2221 include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,35, 40, 45 and 50 mg/kg. These dosages may be combined with rhGAA, suchas ATB-200 rhGAA, at a molar ratio of AT2221 to rhGAA ranging from 15:1to 150:1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60:1, 65:1,70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 125:1, and 150:1. rhGAA and AT2221may be coadministered in these amounts or molar ratios eitherconcurrently, sequentially or separately. The ranges above include allintermediate subranges and values, such as all integer values betweenthe range endpoints.

Representative dosages of AT2220 range from 0.1 to 120 mg/kg, preferably0.25 to 60, and most preferably from 0.6 to 15 mg/kg. Specific dosagesof AT2220 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 and 30mg/kg. These dosages may be combined with rhGAA, such as ATB-200 rhGAA,at a molar ratio of AT2220 to rhGAA ranging from 15:1 to 150:1. Specificratios include 15:1, 20:125:1, 50:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1,90:1, 100:1, 125:1, and 150:1. rhGAA and AT2220 may be coadministered inthese amounts or molar ratios either concurrently, sequentially orseparately. The ranges above include all intermediate subranges andvalues, such as all integer values between the range endpoints.

The rhGAA composition of the invention, its fractions or derivatives mayalso be used for metabolizing, degrading, removing or otherwisedecreasing glycogen in tissue, muscle, muscle fiber, muscle cells,lysosomes, organelles, cellular compartments, or cytoplasm. Byadministering the rhGAA composition to a subject, optionally along witha chaperone or a drug that reduces immunological responses to rhGAA.

In another embodiment of its method of use, the rhGAA of the inventionmay be used for modulating lysosomal proliferation, autophagy, orexocytosis in a cell by administering it, its fractions, or derivativesto cells, tissues, or subjects in need of such modulation, optionally incombination with a chaperone or optionally as a conjugate with anothertargeting moiety. Autophagy is a catabolic mechanism that allows a cellto degrade glycogen or other unnecessary or dysfunctional cellularcomponents through the actions of it lysosomes. This method can alsoinvolve systemically or locally administering the GAA composition to asubject in need of treatment.

The rhGAA according to the invention, which is enriched for mono-M6P andbis-M6P, compared to LUMIZYME® and MYOZYME®, and which has favorablepharmacokinetic properties conferred by its glycosylation pattern mayalso be used for treatment of other conditions requiring the breakdownof complex carbohydrates, such as other disorders in which glycogen orother carbohydrates degraded by rhGAA accumulate in the lysosomes orother parts of the cell, such as in the cytoplasm accessible to rhGAA,such as Glycogen storage disease III. It may also be usednon-therapeutic purposes, such as for the production of foods,beverages, chemicals and pharmaceutical products which require breakingdown complex carbohydrates such as starch and glycogen into theirmonomers.

Examples

The following non-limiting Examples exemplify aspects of the invention.

Section I: ATB-200 rhGAA and its PropertiesLimitations of Existing MYOZYME® and LUMIZYME® rhGAA Products

To evaluate the ability of the rhGAA in MYOZYME® and LUMIZYME®, the onlycurrently approved treatments for Pompe disease, these rhGAApreparations were injected onto a CIMPR column (which binds rhGAA havingM6P groups) and subsequently eluted with a free M6 gradient. Fractionswere collected in 96-well plate and GAA activity assayed by4MU-a-glucose substrate. The relative amounts of bound and unbound rhGAAwere determined based on GAA activity and reported as the fraction oftotal enzyme.

FIGS. 4A and 4B describe the problems associated with conventional ERTs(MYOZYME® and LUMIZYME®): 73% of the rhGAA in MYOZYME® (FIG. 4B) and 78%of the rhGAA in LUMIZYME® (FIG. 4A) did not bind to the CIMPR, see theleft-most peaks in each figure. Only 27% of the rhGAA in MYOZYME® and22% of the rhGAA in LUMIZYME® contained M6P that can productive targetit to the CIMPR on muscle cells, see FIG. 2 which describes productivedrug targeting and non-productive drug clearance.

An effective dose of MYOZYME® and LUMIZYME® corresponds to the amount ofrhGAA containing M6P which targets the CIMPR on muscle cells. However,most of the rhGAA in these two conventional products does not target theCIMPR receptor on target muscle cells. The administration of aconventional rhGAA where most of the rhGAA is not targeted to musclecells increases the risk of allergic reaction or induction of immunityto the non-targeted rhGAA.

Preparation of CHO Cells Producing ATB-200 rhGAA Having a High Contentof Mono- or Bis-M6P-Bearing N-Glycans.

CHO cells were transfected with DNA that expresses rh-GAA followed byselection of transformants producing rhGAA. A DNA construct fortransforming CHO cells with DNA encoding rh-GAA is shown in FIG. 5 . CHOcells were transfected with DNA that expresses rh-GAA followed byselection of transformants producing rhGAA.

After transfection, DG44 CHO (DHFR-) cells containing a stablyintegrated GAA gene were selected with hypoxanthine/thymidine deficient(-HT) medium). Amplification of GAA expression in these cells wasinduced by methotrexate treatment (MTX, 500 nM). Cell pools thatexpressed high amounts of GAA were identified by GAA enzyme activityassays and were used to establish individual clones producing rhGAA.Individual clones were generated on semisolid media plates, picked byCLONEPIX™ system, and were transferred to 24-deep well plates. Theindividual clones were assayed for GAA enzyme activity to identifyclones expressing a high level of GAA. Conditioned media for determiningGAA activity used a 4-MU-a-Glucosidase substrate. Clones producinghigher levels of GAA as measured by GAA enzyme assays were furtherevaluated for viability, ability to grow, GAA productivity, N-glycanstructure and stable protein expression. CHO cell lines, including CHOcell line GA-ATB-200, expressing rhGAA with enhanced mono-M6P or bis-M6PN-glycans were isolated using this procedure.

Purification of rhGAA ATB-200 rhGAA

Multiple batches of the rhGAA according to the invention were producedin shake flasks and in perfusion bioreactors using CHO cell lineGA-ATB-200 and CIMPR binding was measured. Similar CIMPR receptorbinding (˜70%) to that shown in FIG. 6B and FIG. 7 was observed forpurified ATB-200 rhGAA from different production batches indicating thatATB-200 rhGAA can be consistently produced. As shown by FIGS. 6A and 6B,LUMIZYME® rhGAA exhibited significantly less CIMPR binding than ATB-200rhGAA.

Analytical Comparison of ATB-200 to LUMIZYME®

Weak anion exchange (“WAX”) liquid chromatography was used tofractionate ATB-200 rhGAA according to terminal phosphate. Elutionprofiles were generated by eluting the ERT with increasing amount ofsalt. The profiles were monitored by UV (A280 nm). ATB-200 rhGAA wasobtained from CHO cells and purified. LUMIZYME® was obtained from acommercial source. LUMIZYME® exhibited a high peak on the left of itselution profile. ATB-200 rhGAA exhibited four prominent peaks eluting tothe right of LUMIZYME® (FIG. 8 ). This confirms that ATB-200 rhGAA wasphosphorylated to a greater extent than LUMIZYME® since this evaluationis by terminal charge rather than CIMPR affinity.

Oligosaccharide Characterization of ATB-200 rhGAA

Purified ATB-200 rhGAA and LUMIZYME® glycans were evaluated by MALDI-TOFto determine the individual glycan structures found on each ERT (FIG. 9). ATB-200 samples were found to contain slightly lower amounts ofnon-phosphorylated high-mannose type N-glycans than LUMIZYME®. Thehigher content of M6P glycans in ATB-200 than in LUMIZYME®, targetsATB-200 rhGAA to muscle cells more effectively. The high percentage ofmono-phosphorylated and bis-phosphorylated structures determined byMALDI agree with the CIMPR profiles which illustrated significantlygreater binding of ATB-200 to the CIMPR receptor. N-glycan analysis viaMALDI-TOF mass spectrometry confirmed that on average each ATB200molecule contains at least one natural bis-M6P N-glycan structure. Thishigher bis-M6P N-glycan content on ATB-200 rhGAA directly correlatedwith high-affinity binding to CIMPR in M6P receptor plate binding assays(K_(D) about 2-4 nM) (FIG. 10A).

Characterization of CIMPR Affinity of ATB-200

In addition to having a greater percentage of rhGAA that can bind to theCIMPR, it is important to understand the quality of that interaction.LUMIZYME® and ATB200 rhGAA receptor binding was determined using a CIMPRplate binding assay. Briefly, CIMPR-coated plates were used to captureGAA. Varying concentrations of rhGAA were applied to the immobilizedreceptor and unbound rhGAA was washed off. The amount of remaining rhGAAwas determined by GAA activity. As shown by FIG. 10A, ATB-200 rhGAAbound to CIMPR significantly better than LUMIZYME®.

FIG. 10B shows the relative content of bis-M6P glycans in LUMIZYME®, aconventional rhGAA, and ATB-200 according to the invention. ForLUMIZYME® there is on average only 10% of molecules have abis-phosphorylated glycan. Contrast this with ATB-200 where on averageevery rhGAA molecule has at least one bis-phosphorylated glycan.

ATB-200 rhGAA was More Efficiently Internalized by Fibroblast thanLUMIZYME®

The relative cellular uptake of ATB-200 and LUMIZYME® rhGAA werecompared using normal and Pompe fibroblast cell lines. Comparisonsinvolved 5-100 nM of ATB-200 rhGAA according to the invention with10-500 nM conventional rhGAA TRIS base and cells were washed 3-timeswith PBS prior to harvest. Internalized GAA measured by 4MU-a-Glucosidehydrolysis and was graphed relative to total cellular protein and theresults appear in FIG. 11 .

ATB-200 rhGAA was also shown to be efficiently internalized into cells(FIGS. 11A and 11B, respectively), show that ATB-200 rhGAA isinternalized into both normal and Pompe fibroblast cells and that it isinternalized to a greater degree than conventional LUMIZYME® rhGAA.ATB-200 rhGAA saturates cellular receptors at about 20 nM, while about250 nM of LUMIZYME® is needed. The uptake efficiency constant(K_(uptake)) extrapolated from these results is 2-3 nm for ATB-200 and56 nM for LUMIZYME® as shown by FIG. 11C. These results suggest thatATB-200 rhGAA is a well-targeted treatment for Pompe disease.

Section II: Preclinical Studies

ATB-200 rhGAA with Superior Glycosylation was Significantly Better thanStandard of Care ERT for Glycogen Clearance in Skeletal Muscles of GAAKO Mice

As explained above, enzyme replacement therapy (ERT) using recombinanthuman GAA (rhGAA) is the only approved treatment available for Pompedisease. This ERT requires the specialized carbohydrate mannose6-phosphate (M6P) for cellular uptake and subsequent delivery tolysosomes via cell surface cation-independent M6P receptors (CIMPRs).However, the current rhGAA ERT contains low amounts of M6P that limitdrug targeting and efficacy in disease-relevant tissues. The inventorsdeveloped a production cell line and manufacturing process that yieldrhGAA (designated as ATB-200 rhGAA) with superior glycosylation andhigher M6P content than conventional rhGAA, particularly thehigh-affinity bis-M6P N-glycan structure, for improved drug targeting.ATB-200 rhGAA binds the CI-MPR with high affinity (KD˜2-4 nM) and wasefficiently internalized by Pompe fibroblasts and skeletal musclemyoblasts (K_(uptake)˜7-14 nM).

ATB-200 rhGAA clears glycogen significantly better than LUMIZYME® inskeletal muscle. The effects of administering LUMIZYME® and ATB-200rhGAA for glycogen clearance in GAA KO mice were evaluated. Animals weregiven two IV bolus administrations (every other week); tissues wereharvested two weeks after the last dose and analyzed for GAA activityand glycogen content (FIG. 12 ). ATB-200 rhGAA and LUMIZYME® rhGAA wereequally effective for clearing glycogen in heart (FIG. 12A). As show inin FIGS. 12B and 12C, ATB-200 rhGAA at 5 mg/kg was equivalent toLUMIZYME® rhGAA at 20 mg/kg for reducing glycogen in skeletal muscles;ATB-200 dosed at 10 and 20 mg/kg was significantly better than LUMIZYME®for clearing glycogen in skeletal muscles.

Rationale for Co-Administration of ATB-200 rhGAA with AT2221 (CHARTTechnology)

A chaperone binds to and stabilizes rhGAA ERT, increases uptake ofactive enzyme into tissues, improves tolerability and potentiallymitigates immunogenicity. As shown above, the protein stability of ERTunder unfavorable conditions was substantially improved using CHART™(CHART: chaperone-advanced replacement therapy). As shown by FIGS. 13Aand 13B, the stability of ATB-200 was significantly improved by AT2221(Miglustat, N-butyl-deoxynojirimycin). Folding of rhGAA protein wasmonitored at 37° C. by thermal denaturation in neutral (pH 7.4—plasmaenvironment) or acidic (pH 5.2—lysosomal environment) buffers. AT2220stabilized rhGAA protein in neutral pH buffer over 24 hours.

Co-Administration of LUMIZYME® with AT2221 (Miglustat) Compared toCo-Administration of ATB-200 rhGAA with Miglustat

Twelve week old GAA KO mice treated with LUMIZYME® or ATB200, 20 mg/kgIV every other week 4 injections; Miglustat was co-administered at 10mg/kg PO, 30 min prior to rhGAA as indicated. Tissues were collected 14days after last enzyme dose for glycogen measurement. FIG. 14 shows therelative reduction of glycogen in quadriceps and triceps skeletalmuscle.

Reduction of Tissue Glycogen with ATB-200 rhGAA Coadministered withPharmacological Chaperone AT2221 (Miglustat).

The combination of a pharmacological chaperone and ATB-200 rhGAA wasfound to enhance glycogen clearance in vivo. GAA KO mice were given twoIV bolus administrations of rhGAA at 20 mg/kg every other week. Thepharmacological chaperone AT2221 was orally administered 30 mins priorto rhGAA at dosages of 0, 1, 2 and 10 mg/kg. Tissues were harvested twoweeks after the last dose of ERT and analyzed for GAA activity, glycogencontent cell specific glycogen and lysosome proliferation.

As shown by FIG. 15 , the animals receiving ATB200+ chaperone AT2221exhibited enhanced glycogen clearance from quadriceps muscle. ATB-200rhGAA (20 mg/kg) reduced glycogen more than the same dose of LUMIZYME®and when ATB-200 rhGAA was combined with 10 mg/kg of AT2220 near normallevels of glycogen in muscle were attained.

As shown by FIGS. 16A and 16B, unlike conventional rhGAA, which showedlimited glycogen reduction (indicated by abundant punctate PAS signal),ATB-200 rhGAA alone showed a significant decrease in PAS signals.Co-administration with 10 mg/kg miglustat resulted in a substantialfurther reduction in substrate. TEM revealed that the majority ofglycogen in the lysosomes as membrane-bound, electron-dense material,which correspond to the punctate PAS signals. Co-administration ofATB-200 rhGAA with miglustat, reduced the number, size and density ofsubstrate-containing lysosomes suggesting targeted delivery of ATB-200rhGAA to the muscle cells and subsequent delivery to the lysosomes.

From the study (2 IV bolus every other week injections) shown above,tissues were processed for lysosomal proliferation using a LAMP 1marker, the up-regulation is another hallmark of Pompe disease. LAMP:lysosome-associated membrane protein. From the study (2 IV bolus EOWinjections) shown above, soleus tissue was processed for LAMP 1 stainingin adjacent sections and type I fiber-specific antibody (NOQ7.5.4D) inadjacent sections (FIGS. 16C and 16D) ATB-200 rhGAA results in a moresubstantial LAMP1 reduction compared to conventional rhGAA, withreductions leading to levels seen in WT animals (FIG. 16C).

In addition, unlike rhGAA, where the effect is mostly restricted to typeI fibers (slow twitch, marked with asterisks), ATB-200 rhGAA also led tosignificant reduction in LAMP1 signals in a fraction of type II (fasttwitch) fibers (arrow heads) (FIG. 16D). Importantly, co-administrationwith miglustat further improved ATB-200-mediated reduction of LAMP1proliferation in the majority of type II fibers (FIGS. 16C and 16D). Asa result, there did not appear to be a significant fiber type-specificdifference in the level of LAMP1 signals. Similar conclusions were drawnfrom quadriceps and diaphragm (data not shown).

In a separate and similarly designed study, the effect of ATB-200±AT2221was examined over a longer term with 4 biweekly IV bolus injections. Inheart, the main glycogen store in the cardiomyocytes was readily clearedby repeat administration of either rhGAA or ATB-200 to levels seen inwild-type (WT) animals (FIG. 17A). However, the substrate in cardiacsmooth muscle cells seems to be cleared preferably by ATB-200 rhGAA,suggesting a potentially broader bio-distribution of ATB-200 compared torhGAA (asterisks mark the lumen of cardiac blood vessels). Importantly,co-administration with miglustat further improved ATB-200-mediatedreduction of LAMP1 proliferation.

These results show that ATB-200 rhGAA, which has higher levels of M6Pand bis-M6P on its N-glycans efficiently targets CIMPR in skeletalmuscle. ATB-200 rhGAA also has well-processed complex-type N-glycansthat minimize non-productive clearance in vivo, has pharmacokineticproperties favorable for its use in vivo and exhibits good targeting tokey muscle tissues in vivo. They also show that ATB-200 rhGAA is betterthan the conventional standard of care, LUMIZYME®, for reducing glycogenin muscle tissue and that a combination of ATB-200 rhGAA and chaperoneAT2221 further improve removal of glycogen from target tissues andimproves muscle pathology.

1. A composition comprising recombinant human acid alpha-glucosidase(rhGAA), wherein 40%-60% of the N-glycans on the rhGAA are complex typeN-glycans, and wherein the rhGAA comprises at least 3.0 molmannose-6-phosphate (M6P) residues per mol rhGAA.
 2. The composition ofclaim 1, wherein the rhGAA comprises at least 4.0 mol M6P residues permol rhGAA.
 3. The composition of claim 1, wherein the rhGAA comprises atleast 5.0 mol M6P residues per mol rhGAA.
 4. The composition of claim 1,wherein the rhGAA comprises at least 7.0 mol M6P residues per mol rhGAA.5. The composition of claim 1, wherein the rhGAA comprises from 3.0 molto 7.0 mol M6P residues per mol rhGAA.
 6. A composition comprisingrhGAA, wherein 40%-60% of the N-glycans on the rhGAA are complex typeN-glycans, and wherein at least 2% of the total glycans on the rhGAA arebis-phosphorylated mannose-6-phosphate (bis-M6P) glycans.
 7. Thecomposition of claim 6, wherein at least 2.5% of the total glycans onthe rhGAA are bis-M6P glycans.
 8. The composition of claim 6, wherein atleast 3.0% of the total glycans on the rhGAA are bis-M6P glycans.
 9. Thecomposition of claim 6, wherein at least 17% of the total glycans on therhGAA are bis-M6P glycans.
 10. (canceled)
 11. A composition comprisingrecombinant human acid alpha-glucosidase (rhGAA), wherein the rhGAAcomprises at least 6.0 mol M6P residues per mol rhGAA.
 12. Thecomposition of claim 11, wherein the rhGAA comprises at least 7.0 molM6P residues per mol rhGAA.